Summary Our long-term goal is to understand the molecular mechanism underlying mitochondrial biogenesis. Mitochondria produce the majority of cells? adenosine triphosphate (ATP) to be used as ?energy currency? in eukaryotic cells. Apart from their metabolic function, mitochondria participate in a diverse array of (patho)physiological processes such as apoptosis, cancer, degenerative diseases, and aging. The Hap2/3/4/5 complex is a master transcriptional activator of mitochondrial biogenesis in yeast. Hap4 is the regulatory subunit of this complex and its levels determine the activity of the complex. Our preliminary data indicate that mitochondrial biogenesis in yeast is mediated through transcriptional and post-translational regulation of Hap4. We found that Hap4 turnover is mediated through the ubiquitin proteasome system and requires two ubiquitin-conjugating enzymes, Ubc1 and Ubc4. The cysteine residue 22 of Ubc4 is highly conserved among its orthologs and we found that it is required for both a reducing agent- sensitive modification of Ubc4 and Hap4 turnover. We also identified seven separate cis-acting elements in the promoter of HAP4 that are important for HAP4 expression. In this proposal, we are going to achieve the following two aims: 1) To characterize the oxidative modification to Ubc4 and its potential role in achieving cellular redox balance. We are going to purify Ubc4 and determine its oxidative modification via Mass Spectrometry. Elucidation of Ubc4 modification will shed lights on how cells achieve redox homeostasis by regulating mitochondrial biogenesis. 2) To characterize how the HAP4 promoter and trans-acting regulatory factors work together to achieve optimal HAP4 expression and mitochondrial homeostasis. Using a combination of biochemical, genetic, and cell biological tools, we will determine the mechanisms by which mitochondrial biogenesis is regulated by the cellular energetic demand, the Mediator complex, and the functional state of mitochondria. Insights into how cells achieve functional homeostasis of mitochondria may lead to better medical interventions to treat mitochondrial diseases.